CARDIOVIRAL PROTEASES AND GENOME STRUCTURE

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The picornavirus have evolved robust and effective mechanisms to express their proteins. Novel internal ribosomal entry sites (IRESes) allow the genomes to bypass normal translational requirements for 5' cap structures, and efficiently lure the ribosomes into viral instead of cellular pathways. The captured ribosomes pass down a single, long ORF, creating polyproteins that are in reality, tandem linkages of all structural and enzymatic units necessary for infection. The individual protein fragments are liberated co-translationally and post- translationally in a proteolytic cascade that is a defining feature of this family. No fewer than three viral encoded catalytic entities are required for complete processing, none of which has an exact cellular analogue. It is clear that all aspects of the viral life cycle are influenced if not determined by the rates at which the cleavage sites are processed by these enzymes and autocatalytic mechanisms. The in vivo nuances of context and structure are known to impose critical regulatory roles governing the orderly release of proteins throughout the infectious cycle. Impingement on these pathways is invariably fatal, though sometimes for reasons not anticipated from study of mature viral protein or RNA. This program focuses on the murine cardioviruses, encephalomyocarditis virus (EMCV) and Mengovirus. The special propensity of cardiovirus RNAs to facilitate efficient translation in cell-free extracts and the remarkable avidity of the processing cascade during these reactions, are hallmarks of these genomes, and make these isolates exceptionally useful experimental subjects for molecular dissection of the picornavirus life cycle. The goals of this investigation are to explore and define the relationship of the cardiovirus genus to other members of the picornavirus family, and to exploit the unique features of cardioviruses to examine fundamental molecular questions about picornaviral translation, proteolytic processing and morphogenesis. The specific aims are: (1) to probe the translational consequences of synthetic and natural leader protein mutations one IRES-dependent protein expression in vivo. (2) To evaluate the role of defective 2A sequences and "pseudo" primary cleavage reactions in the lethal abrogation of capsid region processing pathways. (3) To explore the requirements for 3C catalyzed processing events in the VPg-dependent initiation of RNA synthesis. (4) To map and define the sequence and structural elements within the cardioviral 3'UTR that are required for translation, replication and infectivity.